Immunoblotting or “Western” blotting technology has been developed to detect specific proteins in a specimen. Western blotting typically includes at least four primary steps including protein separation, protein transfer, membrane blocking, and protein detection. In immunoblotting gel electrophoresis is used to separate native or denatured proteins. For example, one mode of separation using gel electrophoresis separates proteins based on molecular weight. Persons having ordinary skill in the art are aware of the various ways gel electrophoresis may be used to separate proteins, so these variations will not be discussed in detail here.
After protein separation in a western blotting procedure, the separated proteins are typically transferred to a membrane using techniques known to person having ordinary skill in the art. Other steps such as “blocking” may be necessary to ensure that only certain proteins react with the transfer membrane which, in turn, better ensures that detection information is more accurate. The detection step includes probing the membrane for a particular protein of interest using a modified antibody. When the protein of interest is detected by the modified antibody, a reaction takes place that may be used to generate a visual indication. The magnitude of the visual indication corresponds to the amount of protein present. The location of the visual indication in the gel corresponds, for example, to the molecular weight of the protein.
In all experiments using some form of the scientific method, a control group is necessary to verify and/or frame the results obtained with regard to an experimental group. In the case of western blotting, a sample containing unknown proteins is used as the experimental group while samples of known purified proteins are used as the control group to compare to results from the experimental group. In order for a protein to be used as a positive control, however, it must be water soluble and highly purified or the experiment will necessarily have a greater potential of leading to less accurate conclusions.
A positive control is available for many classes of proteins, but no positive control is currently available for any of the membrane-bound glucose transporter proteins such as, for example, SEQ ID NO. 10 (GLUT1), SEQ ID NO. 12 (GLUT4), SEQ ID NO. 13 (GLUT5), and SEQ ID NO. 16 (GLUT12). This is true because all of these proteins are very hydrophobic and are very difficult to purify.
What is needed, therefore, are positive controls for membrane-bound glucose transporter proteins and a method for preparing such positive controls.